Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7 - PubMed (original) (raw)

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7

Jongdae Lee et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor alpha and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-kappaB. However, TLR8 activation by R-848 and TLR2 activation by [S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)] were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

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Figures

Fig. 1.

Fig. 1.

Guanosine and examples of immunostimulatory analogs. 7-deazaG, 7-deazaguanosine; loxoribine, 7-allyl-8-oxoguanosine.

Fig. 2.

Fig. 2.

Guanosine analogs activate murine splenocytes. (A) TOG activates splenocytes. Splenocytes isolated from C57BL/6 mice were incubated with TOG at the indicated concentration for 24 h, and the induction of cytokines was measured by ELISA as described in Materials and Methods. (B) Other guanosine analogs activate splenocytes. Splenocytes were stimulated for 24 h with TOG, loxoribine (Lox), 7-deaza-G, 7-deaza-dG, and 9-hexyl-guanine (100 μM each), and the induction of IL-6 and IL-12(p40) was measured by ELISA. Results are representative of more than three separate experiments, and standard error was determined from duplicates.

Fig. 3.

Fig. 3.

TOG activates murine macrophages and dendritic cells. (A) TOG activates macrophages. BMDMs from C57BL/6 mice were stimulated for 24 h with TOG (100 μM), pam3Cys (5 μg/ml), p(I:C) (5 μg/ml), LPS (10 ng/ml), R-848 (1 μM), or CpG (5 μg/ml). The production of IL-6 and IL-12(p40) was measured by ELISA. (B) TOG activates dendritic cells. BMDDCs from C57BL/6 mice were stimulated for 24 h with TOG (100 μM), LPS (10 ng/ml), R-848 (1 μM), or CpG (5 μg/ml). The induction of cell surface markers was measured by flow cytometry.

Fig. 4.

Fig. 4.

Guanosine analogs activate human PBLs. (A) Guanosine analogs induce TNF-α, IL-12, and IFN-α in human PBLs. Human PBLs were stimulated for 24 h with TOG, loxoribine (Loxo), 7-deaza-G (100 μM each), R-848 (0.5 μM), p(I:C) (5 μg/ml), LPS (10 ng/ml), or CpG (5 μg/ml). The induction of cytokines was measured by ELISA as described in Materials and Methods.(B) Guanosine analogs induce IFN-β in human PBLs. PBLs were stimulated for the indicated time periods with TOG (100 μM), 7-deaza-G (100 μM), 7-deaza-dG (100 μM), loxoribine (Loxo, 100 μM), R-848 (1 μM), LPS (10 ng/ml), or CpG (5 μg/ml), and the induction of IFN-β was measured by RT-PCR as described in Materials and Methods.

Fig. 5.

Fig. 5.

Guanosine analogs activate TLR7. HEK293 cells were transfected with either empty vector or the indicated human TLR. Twenty-four hours after transfection cells were stimulated for 6 h with TNF-α (10 ng/ml), heat-killed Staphylococcus aureus (HKS, 1 × 107/ml), p(I:C) (10 μg/ml), loxoribine (200 μM), TOG (200 μM), R-848 (20 μM), or CpG (5 μM). Relative luciferase activities were measured as described in Materials and Methods.

Fig. 6.

Fig. 6.

TOG and imiquimod activate TLR7 but not TLR8. HEK293 cells were transfected with empty vector, TLR7, or TLR8 and stimulated with TOG (200 μM), imiquimod (20 ng), or R-848 (20 μM) for 6 h. Relative luciferase activities were measured as described in Materials and Methods.

Fig. 7.

Fig. 7.

Activation of TLR7 requires endosomal maturation. (A) Activation of Ramos cells by TOG or R-848 is inhibited by chloroquine. Ramos cells were pretreated with or without chloroquine (5 μM) for 30 min and stimulated with TOG (100 μM), R-848 (1 μM), CpG (5 μg), or pam3Cys (pam3, 5 μg) for 2 h, and activation of NF-κB was measured by electrophoretic mobility-shift assay. (B) Activation of TLR7 but not TLR8 is inhibited by chloroquine. HEK293 cells were transfected with TLR7, TLR8, or TLR9 and stimulated with TOG (200 μM), R-848 (20 μM), or CpG (5 μM) for 6 h. Relative luciferase activities were measured as described in Materials and Methods.

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References

    1. Means, T. K., Golenbock, D. T. & Fenton, M. J. (2000) Life Sci. 68, 241–258. - PubMed
    1. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Huffel, C. V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., et al. (1998) Science 282, 2085–2088. - PubMed
    1. Akira, S. & Hemmi, H. (2003) Immunol. Lett. 85, 85–95. - PubMed
    1. Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. (2001) Nature 413, 732–738. - PubMed
    1. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., Eng, J. K., Akira, S., Underhill, D. M. & Aderem, A. (2001) Nature 410, 1099–1103. - PubMed

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